Optical microscopy is limited, by the wavelength of light, to resolutions in the range of a hundred, and usually hundreds, of nanometer. Scanning electron microscopes (SEMs) do not have this limitation and are able to attain a considerably higher resolution, in the range of a few nanometers.
One of the disadvantages of SEMs is that samples have to be maintained in vacuum, precluding the study of in-vivo processes or the study of wet materials. Furthermore, electrically insulating samples composed of organic materials require coating to avoid charge accumulation.
As early as 1960 a thesis by Thornley (University of Cambridge, 1960) disclosed unsuccessful attempts to maintain a sample intended for electron microscopy in an atmosphere of water vapor. A membrane is used to seal a chamber from the vacuum of the electron beam and the chamber itself has an inlet from a source of water vapor.
Attempts to use Electron Microscopy for living specimens go back as far as 1970. A paper by Swift and Brown (J. Phys. E: Sci. Instrum. 3, 924, 1970) disclosed the use of transmission electron microscopy (TEM) for examination of specimens at atmospheric pressure, for example in water. A cell having an aperture sealed with a collodion-carbon film is used to mount a sample. An electron beam passes through the aperture to strike the sample, and electrons not stopped by the sample continue to a scintillator where photons are produced. At atmospheric pressure the results were found to be “rather noisy” although a resolution of 0.1 μm was claimed.
U.S. Pat. No. 4,071,766 describes an attempt to use electron microscopy to see material in a non-vacuum environment, and refers to the inspection of living objects as “an ever-recurring problem”. U.S. Pat. No. 4,720,633 describes a further attempt to use electron microscopy to see material in a non-vacuum environment. In both of these patents the electron beam travels through an aperture to a wet specimen. Neither of these attempts succeeds, however, in successfully viewing wet objects. The contents of both of these documents are hereby incorporated by reference.
A commercial product which attempts to solve the above problem is an Environmental Scanning Electron Microscope (ESEM), commercially available from Philips Electron Optics of Eindhoven, The Netherlands, which maintains a vacuum gradient along the electron beam path. However, the ESEM requires working with a sample at a critical point of water-vapor equilibrium, and requires cooling of the sample to around 4° C. Inspection of specimens at pressures of up to 5 Torr is said to be possible. However, so far there is no evidence that wet and/or living objects can be viewed at resolutions of 10 nm and below. Further information on this product and how it works can be found in U.S. Pat. Nos. 5,250,808, 5,362,964, and 5,412,211, the contents of which are hereby incorporated by reference.
A common method of achieving high resolution inspection of organic matter is Transmission Electron Microscopy (TEM). TEM requires specially prepared specimens having typical thicknesses in the range of 50 nm. A very high voltage is applied to create a parallel beam that passes through the sample. U.S. Pat. No. 5,406,087, the contents of which are hereby incorporated by reference, discloses a specimen holding device for use with a TEM. A specimen is sealed between two films that are able to transmit an electron beam. The interior of the device is filled with moisture and may be placed within the vacuum environment of the TEM. A very high energy beam travels through the specimen and surrounding fluid leading to a poor signal to noise ratio, as well as considerable damage to the sample.
The information made available by EM is usually unavailable by other techniques (reviewed in Griffiths (2001) Trends in Cell Biology, 11:4:153–154). The main reason for the prevalent underutilization of EM is the complexity of sample preparation, that is not only labor intensive and time consuming, but also raises concerns regarding the biological relevance of the results. The ability to carry out EM in an aqueous environment would obviate these problematic sample preparation steps.
At present, therefore, despite a long-felt need, there is no microscope that permits the study of wet samples at resolutions showing macro-molecular and molecular levels of detail. Such an ability is needed in fields as diverse as cell biology and polymer science as well as industries such as petroleum, food and microelectronics. In particular in the field of cell biology, such a microscope would enable analysis of cells leading to measurements of molecular level processes and also opening a whole new field in pharmaceutical drug discovery and diagnostic measurement. For example, such a microscope would allow detailed study and direct observation of interactions between drugs and living cells.